摘要
研究核苷类抗病毒药物齐多夫定(zidovudine,AZT)对小鼠整体代谢及肝脏糖脂代谢平衡的影响。雄性ICR小鼠连续灌胃齐多夫定12周,每天记录小鼠的饮水量及摄食量。检测给药12周后不同禁食时间血清葡萄糖(GLU)、甘油三酯(TG)水平以及血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平,进行糖耐量实验(OGTT)和胰岛素耐量实验(ITT);HE染色观察肝脏病理学变化;RT-PCR检测葡萄糖转运蛋白(Glut2)、肉碱棕榈酸转移酶(Cpt1α)、中链酰基辅酶A脱氢酶(Mcad)以及磷酸烯醇式丙酮酸羧激酶(Pepck)、葡萄糖-6-磷酸酶(G6pase)的基因水平;Western blot检测肝脏胰岛素信号Akt、P-Akt及Glut2、Mcad、Cpt1α的蛋白水平。结果显示,齐多夫定导致禁食后脂代谢能力显著下降,糖耐量受损,肝细胞体积增大,显著增加肝脏TG、非酯化脂肪酸(NEFA)含量,提高Glut2基因表达,下调脂肪酸氧化代谢基因Cpt1α、Mcad和糖异生相关基因水平,以及下调Cpt1α的蛋白表达。实验结果提示齐多夫定能够引起禁食后糖脂代谢紊乱,且呈一定的剂量依赖性。
艾滋病,即获得性免疫缺陷综合征(AIDS),是世界范围内的重大感染性疾病,感染人数还在不断上
齐多夫定(AZT)是第一个被批准用于治疗AIDS的核苷类逆转录酶抑制
正常情况下,机体组织细胞可以利用葡萄糖、脂肪酸等多种能量物质代谢供能,可根据组织环境和能量物质的供应情况,在不同能量代谢底物之间切
齐多夫定(CAS:A122924,纯度大于98%,阿拉丁试剂有限公司);胰岛素注射液(诺和灵30R,丹麦诺和诺德公司);RIPA裂解液、BCA蛋白浓度测定试剂盒(上海碧云天生物技术研究所);总RNA提取试剂,HiScrip
Legend Micro 21R低温离心机,Varioskan Lux多功能微孔板读数仪,Nanodrop ND-2000超微量核酸蛋白测定仪,Applied Biosystems StepOn
24只雄性ICR小鼠随机分为3组,每组8只,分别为溶剂对照组(CON)、齐多夫定低、高剂量组(AZT-L、AZT-H)。齐多夫定给药组每天灌胃给予AZT 100,300 mg/kg,溶剂对照组灌胃给予相应体积的蒸馏水,连续12周。给药结束后禁食12 h摘眼球采血,颈椎脱臼处死小鼠,分离肝脏。
12周给药结束后,分别在禁食6 h和12 h后进行眼眶取血,将全血于200 μL离心管中常温静置30 min后,4 ℃,3 000 r/min离心15 min,吸取上层血清至200 μL EP管中,按血生化试剂盒说明测定血清葡萄糖(GLU)和甘油三酯(TG)水平。
给药结束前7天,小鼠禁食不禁水12 h后,灌胃给予2 g/kg葡萄糖溶液。分别测定给予葡萄糖0,15,30,60,120 min后小鼠的血糖,计算血糖浓度-时间曲线下面积(AUC)。
小鼠肝脏用生理盐水漂洗干净,滤纸吸干称重,计算肝脏指数,即肝脏占体重的质量分数。称取肝组织30 mg,加入磷酸盐缓冲液(PBS)研磨,使用BCA法测定蛋白浓度,吸取肝匀浆上清液测定肝脏组织TG、非酯化脂肪酸(NEFA)水平。
称取肝组织30 mg,加入RIPA裂解液提取肝总蛋白,使用BCA法定蛋白,金属浴煮蛋白,-20 ℃保存变性蛋白。取等体积蛋白进行SDS-PAGE凝胶电泳,在冰上恒流模式下进行转膜,5% BSA封闭2 h,4 ℃孵育一抗过夜,一抗为Cpt1α、Mcad、Glut2、Akt、P-Akt抗体。第2天用TBST洗膜,孵育二抗羊抗兔IgG抗体1 h,再洗膜,采用化学发光法曝光。使用Image J进行蛋白条带的灰度分析。
称取肝组织30 mg,加Trizol裂解液1 mL提取总RNA。使用超微量核酸蛋白测定仪检测RNA浓度,在逆转录仪上将RNA逆转录成cDNA。运用实时荧光定量PCR法测定cDNA样品中的Cpt1α、Mcad、Pepck、G6pase和Glut2的基因水平。选用β-actin作为内参基因,引物序列如
如

Figure 1 Effect of zidovudine (AZT) on the body weight (A) and liver index (B) in mice ()
CON: Solvent control; AZT-L: Zidovudine(100 mg/kg); AZT-H: Zidovudine(300 mg/kg)
如

Figure 2 Effect of AZT on serum glucose(A) and triglyceride (TG)(B) in mice after fasting for 6 h or 12 h ()
*P < 0.05
如

Figure 3 Effect of AZT in oral glucose tolerance test (OGTT) (A) and insulin tolerance test (ITT) (B) ()
*P < 0.05,
胰岛素耐量(ITT)结果显示,溶剂对照组小鼠注射胰岛素后,血糖急速下降,在30 min时降到最低,随后90 min内肝脏糖异生增加使血糖恢复至基础水平。与对照组相比,AZT给药组可以剂量依赖性的增加胰岛素注射后30 min内的降糖速率,然而,AZT各剂量组血糖上升趋势明显被抑制,提示小鼠肝脏糖异生能力受损(
肝脏HE染色结果如

Figure 4 Effect of AZT on liver histopathology of mice (HE staining, × 200)
A: CON; B: AZT-L; C: AZT-H
血清生化结果显示,AZT给药组呈剂量相关性的增加ALT、AST水平,说明AZT可导致肝脏受损(

Figure 5 Effect of AZT on liver of mice ()
A: Alanine aminotransferase (ALT);B: Aspartate aminotransferase (AST);C: TG D: Non-esterified fatty acids (NEFA)
PCR结果如

Figure 6 Effects of AZT on gene expression of glycolipid metabolism in mice ()
*P < 0.05,
如

Figure 7 Effect of AZT on the expression of protein relative to glucose and lipid metabolism in mice ()
A: Glut2; B: Mcad; C: Cpt1α; D: P-Akt/Akt
新陈代谢是生命体的基本要素,既有整体的特征,也有组织细胞层面的机制。当疾病、损伤、药物等外界因素作用于机体代谢的某个过程,如果超过了机体自身调节能力,即表现为某些代谢失衡特征,甚至诱发多种代谢性疾
葡萄糖是机体多数组织主要的能量代谢底物,也是多数代谢失衡病症的主要易损指
脂肪酸作为另一种主要的能量物质,可与葡萄糖共同参与能量代谢供应,这即是机体代谢灵活性特征之
线粒体是糖、脂等能量物质转化的主要场所,在糖脂代谢调节中发挥关键作
总之,AZT长期给药可导致小鼠代谢失衡,主要表现为糖耐量受损和脂肪酸代谢障碍,肝脏是其重要的靶器官,其可能作用机制是通过下调脂肪酸氧化代谢及糖异生基因表达,从而导致肝脏脂质蓄积和糖脂代谢紊乱。机体糖脂代谢的调节是由多器官参与的,器官之间的交叉对话在能量稳态调节中也至关重要,后续研究可继续从代谢平衡调节着手,通过肌肉、脂肪、胰腺等多器官探讨,进一步阐释AZT导致代谢紊乱的机制。
References
Chen XZ,Wang R,Zhang YJ,et al. Changes in epidemiological profiles of AIDS in China:a systematic analysis[J]. Lancet,2019,394:S8. [百度学术]
AIDS and Hepatitis C Professional Group,Society of Infectious Diseases,Chinese Medical Association. Chinese guidelines for diagnosis and treatment of HIV/AIDS (2021 edition)[J]. Chin J Intern Med(中华内科杂志),2021,60(12):1106-1128. [百度学术]
Sapuła M,Suchacz M,Załęski A,et al. Impact of combined antiretroviral therapy on metabolic syndrome components in adult people living with HIV:a literature review[J]. Viruses,2022,14(1):122. [百度学术]
Tsai FJ,Ho MW,Lai CH,et al. Evaluation of oral antiretroviral drugs in mice with metabolic and neurologic complications[J]. Front Pharmacol,2018,9:1004. [百度学术]
Yarchoan R,Klecker RW,Weinhold KJ,et al. Administration of 3'-azido-3'-deoxythymidine,an inhibitor of HTLV-III/LAV replication,to patients with AIDS or AIDS-related complex[J]. Lancet,1986,1(8481):575-580. [百度学术]
Clumeck N,de Wit S. Update on highly active antiretroviral therapy:progress and strategies[J]. Biomed Pharmacother,2000,54(1):7-12. [百度学术]
Günthard HF,Saag MS,Benson CA,et al. Antiretroviral drugs for treatment and prevention of HIV infection in adults:2016 recommendations of the international antiviral society-USA panel[J]. JAMA,2016,316(2):191-210. [百度学术]
Igoudjil A,Massart J,Begriche K,et al. High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes[J]. Toxicol In Vitro,2008,22(4):887-898. [百度学术]
Lin H,Stankov MV,Hegermann J,et al. Zidovudine-mediated autophagy inhibition enhances mitochondrial toxicity in muscle cells[J]. Antimicrob Agents Chemother,2018,63(1):e01443-e01418. [百度学术]
Santos-Llamas A,Monte MJ,Marin JJG,et al. Dysregulation of autophagy in rat liver with mitochondrial DNA depletion induced by the nucleoside analogue zidovudine[J]. Arch Toxicol,2018,92(6):2109-2118. [百度学术]
Bañó M,Morén C,Barroso S,et al. Mitochondrial toxicogenomics for antiretroviral management:HIV post-exposure prophylaxis in uninfected patients[J]. Front Genet,2020,11:497. [百度学术]
Goodpaster BH,Sparks LM. Metabolic flexibility in health and disease[J]. Cell Metab,2017,25(5):1027-1036. [百度学术]
Morio B,Panthu B,Bassot A,et al. Role of mitochondria in liver metabolic health and diseases[J]. Cell Calcium,2021,94:102336. [百度学术]
Ruocco MR,Avagliano A,Granato G,et al. Metabolic flexibility in melanoma:a potential therapeutic target[J]. Semin Cancer Biol,2019,59:187-207. [百度学术]
Smith RL,Soeters MR,Wüst RCI,et al. Metabolic flexibility as an adaptation to energy resources and requirements in health and disease[J]. Endocr Rev,2018,39(4):489-517. [百度学术]
Lagathu C,Béréziat V,Gorwood J,et al. Metabolic complications affecting adipose tissue,lipid and glucose metabolism associated with HIV antiretroviral treatment[J]. Expert Opin Drug Saf,2019,18(9):829-840. [百度学术]
Xiao JQ,Xu J,Shu FR,et al. Effect of Artemisia Argyi Folium ethanolic extract on blood glucose and blood lipids in diabetic mice[J]. J China Pharm Univ(中国药科大学学报),2021,52(1):71-76. [百度学术]
Soeters MR,Soeters PB,Schooneman MG,et al. Adaptive reciprocity of lipid and glucose metabolism in human short-term starvation[J]. Am J Physiol Endocrinol Metab,2012,303(12):E1397-E1407. [百度学术]
Gambardella J,Lombardi A,Santulli G. Metabolic flexibility of mitochondria plays a key role in balancing glucose and fatty acid metabolism in the diabetic heart[J]. Diabetes,2020,69(10):2054-2057. [百度学术]
Tucci S,Alatibi KI,Wehbe Z. Altered metabolic flexibility in inherited metabolic diseases of mitochondrial fatty acid metabolism[J]. Int J Mol Sci,2021,22(7):3799. [百度学术]
Tomašić N,Kotarsky H,de Oliveira Figueiredo R,et al. Fasting reveals largely intact systemic lipid mobilization mechanisms in respiratory chain complex III deficient mice[J]. Biochim Biophys Acta Mol Basis Dis,2020,1866(1):165573. [百度学术]
Qiu XC,Li J,Lv SH,et al. HDAC5 integrates ER stress and fasting signals to regulate hepatic fatty acid oxidation[J]. J Lipid Res,2018,59(2):330-338. [百度学术]
Bideyan L,Nagari R,Tontonoz P. Hepatic transcriptional responses to fasting and feeding[J]. Genes Dev,2021,35(9/10):635-657. [百度学术]